|
| Status |
Public on Aug 12, 2016 |
| Title |
Genome-scale high-resolution mapping of activating and repressive nucleotides in regulatory regions |
| Organisms |
Escherichia coli; Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
|
| Summary |
We employ a massively parallel reporter assay (MPRA) to measure the ex vivo activities in K562 and HepG2 cells of more than 15,000 human candidate regulatory regions. The regulatory regions were tiled with overlapping constructs enabling high resolution computational inference of activating and repressive nucleotides.
|
| |
|
| Overall design |
Reporter mRNA-seq from HepG2 and K562 cells were transfected with a ~54,000-plex MPRA plasmid pool for a 'PilotDesign' and ~244,000-plex pool for a 'ScaleUpDesign'. In the 'PilotDesign' each regulatory region was associated with constructs at nine positions spaced in 30-bp increments with each position linked to 24 10-nt tags. In the 'ScaleUpDesign' each regulatory region was associated with constructs at 31 positions spaced in 5-bp increments with each position linked to a single 10-nt tag. There were two separate sets of sequences based on the 'ScaleUpDesign'. In the 'PilotDesign' all experiments were conducted with a SV40 promoter, while for the 'ScaleUpDesign' there were separate experiments based on a SV40 and minimal TATA promoter. The reporter mRNA tags facilitate quantitation of their abundances. The same tags were also sequenced from the transfected MPRA plasmid pool to facilitate normalization to plasmid copy numbers. All transfections were done in duplicate. MPRA plasmid pools were amplified and sequenced in replicate for the pilot design and for each design and promoter type for the 'ScaleUpDesign'.
|
| |
|
| Contributor(s) |
Ernst J, Melnikov A, Zhang X, Wang L, Rogov P, Mikkelsen TS, Kellis M |
| Citation(s) |
27701403 |
| Submission date |
Jul 23, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jason Ernst |
| E-mail(s) |
jason.ernst@ucla.edu
|
| Organization name |
UCLA
|
| Department |
Biological Chemistry
|
| Street address |
615 Charles E Young Dr South
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platforms (4)
|
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL14548 |
Illumina HiSeq 2000 (Escherichia coli) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL18133 |
Illumina HiSeq 2500 (Escherichia coli) |
|
| Samples (26)
|
|
| Relations |
| BioProject |
PRJNA290789 |
| SRA |
SRP061466 |
Less...
More...